cDNA Synthesis
In the presence of Mn.sup.++, various DNA-dependent DNA polymerases can act as reverse transcriptases by utilizing RNA as a template and synthesizing a complementary cDNA initiated from an oligonucleotide primer (Karkas, J. D., Proc. Natl. Acad. Sci. USA 70:3834-3838, 1973). The reaction is regarded as inefficient when compared to the rate of synthesis, overall yield of cDNA, and abundance of full-length transcripts produced by bona-fide reverse transcriptases such as avian myeloblastosis virus (AMV) or murine Moloney leukemia virus (MMLV) reverse transcriptases (Powell, L. M., et al., Cell 50:831-840, 1987).
Thermostable DNA-dependant DNA polymerases acting as reverse transcriptases have been used for cDNA synthesis. Reactions carried out at elevated temperatures (greater than 50.degree. C.) are expected to show increased priming specificity and ability to reverse transcribe through regions of RNA secondary structure compared to mesophilic enzyme reactions. Thermostable DNA polymerases that can be used as reverse transcriptases have also been used in the RT-PCR process to create DNA copies (amplicons) starting with an RNA target (Myers, T. W. and Gelfand, D. H. Biochemistry 30:7661-7666, 1991 and U.S. Pat. Nos. 5,322,770; 5,310,652; and 5,407,800).
Betaine
Betaine (N,N,N,-trimethylglycine) has been shown to improve PCR efficiency. U.S. Pat. No. 5,545,539 (Miller, filed Oct. 18, 1994) describes a method of improving the PCR amplification of a target nucleotide sequence by use of an effective amount of a glycine-based osmolyte. The osmolyte reduces the appearance of "stutter bands" in the amplification product, thus allowing for easier detection of the target nucleotide sequence.
Rees et al. (Biochemistry 32:137-144, 1993) demonstrate that betaine has the ability to reduce or eliminate the base pair composition dependence of DNA thermal melting transitions.
DE4411588C1 discloses a buffer for RNA and DNA polymerase reactions containing betaine and the use of the buffer in PCR reactions and in reverse transcription of RNA into cDNA using MMLV reverse transcriptase.
Needed in the art is a method of increasing the overall yield of cDNA and abundance of full-length transcripts in order to improve the sensitivity and quantitative aspects of the overall RT-PCR process.